Electrochemical Genosensing of Overexpressed GAPDH Transcripts in Breast Cancer Exosomes

Exosomes are receiving highlighted attention as new biomarkers for the detection of cancer since they are profusely released by tumor cells in different biological fluids. In this paper, the exosomes are preconcentrated from the serum by immunomagnetic separation (IMS) based on a CD326 receptor as a specific epithelial cancer-related biomarker and detected by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts. Following the lysis of the captured exosomes, the released GAPDH transcripts are amplified by reverse transcription polymerase chain reaction (RT-PCR) with a double-tagging set of primers on poly(dT)-modified-MPs to increase the sensitivity. The double-tagged amplicon is then quantified by electrochemical genosensing. The IMS/double-tagging RT-PCR/electrochemical genosensing approach is first demonstrated for the sensitive detection of exosomes derived from MCF7 breast cancer cells and compared with CTCs in terms of the analytical performance, showing an LOD of 4 × 102 exosomes μL–1. The genosensor was applied to human samples by immunocapturing the exosomes directly from serum from breast cancer patients and showed a higher electrochemical signal (3.3-fold, p < 0.05), when compared with healthy controls, suggesting an overexpression of GAPDH on serum-derived exosomes from breast cancer patients. The detection of GAPDH transcripts is performed from only 1.0 mL of human serum using specific magnetic particles, improving the analytical simplification and avoiding ultracentrifugation steps, demonstrating to be a promising strategy for minimal invasive liquid biopsy.

(5%) atmosphere. Once cells reached approximately 95% confluence on T-175 flask, the culture medium was removed and stored at -21ºC until to exosome isolation.
Exosomes were purified according to Théry et al. 1 with minor changes. The supernatant of the cell culture from MCF7 breast cancer cell line, or from human serum was subjected to differential centrifugation as follow: 300 x g for 10 minutes (removal of residual cells), 2,000 x g for 10 minutes and 10,000 x g for 30 minutes (removal of cellular S4 debris). Then, a Beckman Coulter Optima L-80XP Ultracentrifuge at 100,000 x g for 60 minutes with a 70Ti rotor to pellet exosomes. After that, the supernatant was carefully removed, and crude exosome-containing pellets were resuspended in 1 mL of Tris 1x buffer (pH 7.4, 0.22 µm sterile-filtered) and pooled. A second round of same ultracentrifugation setting was carried out, and the resulting exosome pellet resuspended in 500 µL (per 100 mL of supernatant) of Tris 1x buffer (pH 7.4, 0.22 µm sterile-filtered), and storage at -80°C. All centrifugation steps performed at a temperature of 4°C.
The exosomal protein content was determined by using Pierce BCA Protein Assay Kit (ref. 23227, Thermo Fisher Scientific), following the manufacturer protocol, using bovine serum albumin (BSA) standards in Tris 1x buffer. The spectrophotometric measurements were done at 562 nm.

S3. Immobilization of exosomes and antibodies on magnetic particles
Dynabeads M450 tosylactivated superparamagnetic particles (MPs, 4.5 µm in diameter) has a core of iron oxide salt encapsulated by a polystyrene polymer, which has a polyurethane external layer with the p-toluenesulfonate group 2 . It is a good leaving group, which allows an SN2 reaction to occur in the presence of a nucleophile 3,4 . A nucleophilic reaction by an antibody, protein, peptide, or glycoprotein removes and replaces the sulfonyl ester groups from the polyurethane layer.
Two different approaches were used, as depicted in Figure S1. The first one involves the direct covalent immobilization of exosomes on magnetic particles (Fig. S1, panel A). The second approach is based on the covalent immobilization of the antibodies for a further immunomagnetic separation (IMS) of exosomes (Fig. S1, panel B).
The reaction kinetics are increased by adding 0.1 mol L -1 borate buffer, pH 8.5, in order to ensure the nucleophilic reaction by the amine group. The incubation step was performed overnight with gentle shaking at 4ºC. After that, 0.5 mol L -1 glycine solution was added to ensure the blocking of the any remaining tosylactivated groups, by an incubation for 2 h at 25ºC. After that, the exosomes-modified magnetic particles (exosomes-MP) were resuspended in 160 µL of Tris 1x buffer in order to achieve 1 x 10 6 MPs per 10 µL. The exosomes-MP were maintained at 4ºC until use and remain stable on MPs up to two months.

Immobilization of antibodies on magnetic particles
The CD81 antibody (15 μg mL -1 , as previously optimized 5 ) was added to 55 µL (2.2 x 10 7 MPs) Dynabeads M450 tosylactivated (Fig. S1, panel B). The reaction kinetics are increased by adding 0.1 mol L -1 borate buffer pH 8.5 and 3 mol L -1 ammonium sulphate in borate buffer. The incubation step was performed overnight (18-20h) with gentle shaking at 37ºC. After that, a blocking step with 0.5 mol L -1 glycine solution was performed for 2 h to ensure the blocking of the any remaining tosylactivated groups. After that, the antibody-modified magnetic particles (herein, antiCDX-MPs, where antiCDX = antiCD81) were resuspended in 220 µL (10 µL per assay to give 1 x 10 6 particles per assay) of Tris 1x buffer.
It is important to highlight that in this procedure it was no possible to achieve the immobilization of antiCD326 antibody on MPs. Therefore, commercially modified particles with EpCAM were used. EpCAM corresponds to CD326 (Cluster of Tris 1x containing 0.5% BSA. The same procedure of labeling was performed in the case of the exosomes derived from MCF7 breast cancer cell line, but in this approach, and due to their size and resolution of the technique, the exosomes were firstly immobilized on the surface of MPs, as described on To achieve that, 3.5 x 10 10 exosomes were covalently immobilized on 1.6 x 10 7 MPs, as described in S3, followed by the indirect labeling as described above, with antiCD81 or antiCD326. The same batch of cells and exosomes analyzed by flow cytometry were subjected for confocal microscopy imaging for the study of the binding pattern of antibodies. In the case of cells, the cellular DNA was stained previously (before labeling S7 with antibodies) with Hoechst dye (blue fluorescent dye, excitation 350 nm, emission 490 nm).

Immunomagnetic separation of the cells and exosomes
The IMS of the cells exosomes was performed by antiCDX-MPs (being CDX any of CD81 or CD326 biomarkers) (containing 1 × 10 6 MPs per tube), and 100 µL of MCF-7 cells (concentration ranging from 50 to 5.000 cells mL -1 ) or exosomes (concentration ranging from 100 to 4.0 x 10 4 exosomes µL -1 ), which were simultaneously incubated for 30 min with gentle shaking at 25°C, followed by three washing steps with Tris 1x buffer containing 0.5% BSA. The coated antiCDX-MPs were resuspended in 1.0 mL of Lysis/Binding buffer. Then, they were disrupted by pipetting up and down a couple of times to ensure a complete lysis. In order to ensure sample homogenization, the lysate was passed through a 21-gauge needle using a 2.0 mL syringe. Then, the lysate and antiCDX-MPs were separated by using a magnet plate separator, an antiCDX-MPs pellet on the bottom tube is formed, and the lysate is transferred to another tube.

Double-tagging RT-PCR on magnetic beads
The lysate of the cells or exosomes was incubated with 15 µL of poly(dT)-MPs (7

Optimization of RT-PCR amplification cycles
As aforementioned, the detection of exosomes is a challenging task due to the low concentration in biological samples. Moreover, an intrinsic characteristic of the exosomes is the low RNA content compared to cells 6 . In order to increase the sensitivity of the approach, the double-tagging RT-PCR was optimized towards the number of cycles required for GAPDH transcript detection in exosomes. The cellular GAPDH transcript detection was used for comparison purposes. The double-tagging RT-PCR was performed with 28, 32, 36 and 40 cycles.
The double-tagged amplicons were submitted in parallel to gel electrophoresis and measured by electrochemical magneto genosensing. Negative controls were performed with all reagents, omitting the RNA (from cells and/or exosomes). Figure S2, panel A shows the gel electrophoresis for cellular and exosomal GAPDH amplicons.
While the GAPDH amplicons from MCF7 cells were observed in all PCR cycles tested, the amplicons for exosomes are only evidenced after 36 cycles. Then, the GAPDH amplicons from exosomes were subjected to electrochemical genosensing. As expected, the electrochemical genosensing revealed that in the exosomes the GAPDH transcript was also amplified in all PCR cycles tested (Fig. S2, panel B). However, the signal-tonoise ratio for the detection of the GAPDH amplicons is affected substantially by the increase in the PCR cycles (Fig. S2, panel B inset). Probably, this is due to a larger S9 number of dimers formed as the PCR cycles increases, and the best signal-to-noise ratio was obtained with 32 cycles of PCR, as shown in the inset of the Figure S2, panel B, since higher cycles shows saturation of the magnetic particles with the product. This result also highlights the higher sensitivity of the double-tagging RT-PCR coupled to the electrochemical detection compared with the gel electrophoresis.

Electrochemical magneto-genosensing
The procedure for the detection of the BIO-DIG double-tagged PCR product is based on the immobilization on streptavidin-modified magnetic particles and its electrochemical detection with specific antibody for digoxigenin modified with HRP. The magneto-actuated electrochemical genosensing (Fig. 1, panel C)  For the electrochemical readout, the strep-MPs coated with the amplicons were separated by using a magnet tube separator, a MPs pellet on the bottom tube is formed, followed by remove of the supernatant. Following, MPs pellet is resuspended in ePBS buffer and a magneto-actuated graphite-epoxy composite (m-GEC) electrode is inserted into tube for remove the MPs pellet onto m-GEC surface, which is transferred into an S10 electrochemical cell and measured by means of amperometry at -100 mV vs.
Ag/AgCl/KCl(sat.) by using hydroquinone mediator. For that, a standard one compartment three-electrode electrochemical cell is filling with 19.8 mL of ePBS, 100 μL of 400 mmol L -1 hydroquinone (HQ) as electrochemical mediator, and 100 μL of 400 mmol L -1 H2O2 as substrate. A reproducible steady-current was obtained after 60 s. The cathodic current generated by monitoring benzoquinone species directly related with the amount of captured exosomes. The m-GEC surface cleaning procedure was carried out for every experiment. First, the electrode surface was cleaned with absorbent paper, then by an electrochemical treatment by applying a potential of +3 V for 5 s in 0.5 mol L −1 H2SO4 supporting electrolyte.

S6. RNA integrity analysis and DNA sequencing
A comparative integrity study of RNA from MCF7 breast cancer and purified exosomes was performed. To achieve that, the RNA obtained by lysis of cells and exosomes were processed by classical RNA extraction and purification procedure followed by integrity analysis. The Total Exosome RNA and protein isolation kit were to characterize and quantify the RNA content. Figure S3 shows the results of the RNA integrity analysis. Firstly, the quality of the extracted RNA was assessed by the Bioanalyzer RNA integrity numbers (RIN; 1 = totally degraded, 10 = intact). The cellular 18S and 28S ribosomal RNA (rRNA) are the most dominant peaks, and the RIN value was estimated to be 8.0 consistent with a good RNA quality (Fig. S3, panel A). In addition to the rRNA, one broadband (~100 to ~450 nucleotides) for cellular messenger RNA (mRNA) also is displayed. Unfortunately, rRNA nor mRNA peaks were not observed for RNA extracted from exosomes (Fig. S3, panel   B). Since the algorithm is based on the ribosomal RNA, previous studies demonstrated that exosomes contain little or no rRNA 7,8 and mRNA 9 , RIN values are only valid for cellular RNA quality assessments. It is important to highlight that RNA yield can differ substantially between different RNA isolation methods, which may be related to the low sensitivity of the extraction method. The human serum samples (healthy and breast cancer patients) were separated from the blood cells using a sterile empty tube without any anticoagulant, leave the tube S13 in a standing position for about 20-30 minutes for blood to be clotted. After that, centrifugation at 1500 x g (20 ºC) for 10 minutes was carried out for removal of residual cells and cellular debris. Following, the human serum (supernatant on top) was carefully removed, freeze at -80 ºC to preserve for further assays.

Detection of GAPDH transcripts from purified exosomes without preconcentration on MPs
This approach (Fig. 4, panel A) is based on amplification and detection through non-specific GAPDH biomarker. Firstly, exosomes were isolated from 1.0 mL of human serum from healthy (n = 10, pooled) and breast cancer (n = 10, pooled) patients by ultracentrifugation and resuspended in Tris 1x buffer, as described in S2 (Supp. Data).
Then, the exosomes samples from healthy and breast cancer patients were analyzed with the BCA protein assay kit, and their protein concentrations were estimated to be 235 µg mL -1 and 335 µg mL -1 , respectively. To normalize the results according to the protein content, 0.33 µg of exosomes from healthy and breast cancer patients were subjected to RNA extraction based on poly(dT)-MPs, followed by double-tagging PCR, and subsequent electrochemical genosensing, as described above.
Immunomagnetic separation of the exosomes from undiluted human serum Our detection approach was to isolate and detect exosomes from undiluted human serum (healthy and breast cancer patients) directly by immunomagnetic separation (IMS) based on antiCD326-MPs (Fig. 4, panel B). In this case, samples of undiluted human serum from healthy (n=10, pooled) and breast cancer patients (n=10, pooled) were centrifuged at 10,000 x g to eliminate possible cell debris remaining in the serum. The IMS of the exosomes was performed with antiCD326-MPs (containing 1 × 10 6 MPs per tube), and 1.0 mL of undiluted human serum, incubated for 30 min with gentle shaking at 25 °C, followed by three washing steps with Tris 1x buffer containing 0.5% BSA. Then, the exosomes-coated antiC326-MPs were resuspended with 100 µL of Tris 1x buffer, stored on ice and immediately used for RNA extraction. The exosomescoated antiCD326-MPs were resuspended in 1.0 mL of Lysis/Binding buffer. The exosomes were disrupted by pipetting up and down a couple of times to ensure a complete lysis. In order to ensure sample homogenization, the lysate was passed through a 21-gauge needle using a 2.0 mL syringe. Then, the lysate and antiCD326-MPs were separated by using a magnet plate separator, an antiCD326-MPs pellet on the bottom tube is formed, followed by lysate separation, and transferred by another tube.
finally resuspended with 100 µL of DEPC-treated water. The suspension of RNApoly(dT)-MPs was stored on ice and immediately used for reverse transcription reaction, as for the case of the exosomes derived from MCF7 cells.
The double-tagging polymerase chain reaction (PCR) was also performed as above for the case of the exosomes derived from cell culturing.

Electrochemical magneto-genosensing
The procedure for the detection of the BIO-DIG double-tagged PCR product was also performed as above for the case of the exosomes derived from cell culturing. Figure S4. Panel A shows the control of the purified total exosome population obtained by ultracentrifugation (100,000 x g) normalized according to protein content (0.33 µg per assay). Panel B. Electrochemical genosensing of CD326+ exosomes from 1 mL of cell-free undiluted human serum (centrifuged at 10,000 g) based on immunomagnetic separation with antiCD326-MP and further GAPDH transcripts detection. In all cases, serum-derived exosomes from healthy controls (n = 10, pooled) and breast cancer (n = 10, pooled) patients were processed. The error bars show the standard deviation for n = 3. The raw data for the amperograms are also shown.

S15
To confirm the significance of the differences in the value for the healthy control and breast cancer patient samples, a one-tailed p-test (Hi > Ho) at a 95 % significance level was performed, being Hi hypothesis and Ho the null hypothesis. The mean value and standard deviations of the electrochemical measurements depicted in Figure S4 were used to calculate the p-values of both comparisons, purified exosomes (Fig. S4, panel A) and human serum samples (Fig. S4, panel B). The calculation was done with data analysis tool, considering equal variances in the samples, obtaining the following results: Purified exosomes (Fig. S4, panel A): p = 0.00017 → p<0.05 Human serum samples (Fig. S4,